In solution small-angle neutron scattering has been used to study the solvation properties of lysozyme dissolved in water/glycerol mixtures. To detect the characteristics of the protein-solvent interface, 35 different experimental conditions (i.e., protein concentration, water/glycerol fraction in the solvent, content of deuterated compounds) have been considered and a suitable software has been developed to fit simultaneously the whole set of scattering data. The average composition of the solvent in the close vicinity of the protein surface at each experimental condition has been derived. In all the investigated conditions, glycerol resulted especially excluded from the protein surface, confirming that lysozyme is preferentially hydrated. By considering a thermodynamic hydration model based on an equilibrium exchange between water and glycerol from the solvation layer to the bulk, the preferential binding coefficient and the excess solvation number have been estimated. Results were compared with data previously derived for ribonuclease A in the same mixed solvent: even if the investigated solvent compositions were very different, the agreement between data is noticeable, suggesting that a unique mechanism presides over the preferential hydration process. Moreover, the curve describing the excess solvation number as a function of the solvent composition shows the occurrence of a region of maximal hydration, which probably accounts for the changes in protein stability detected in the presence of cosolvents.

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The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 Â 4.2 Â 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis.The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein.Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.

In this work an improved methodology for studying interactions of proteins in solution by small-angle scattering is presented. Unlike the most common approach, where the protein-protein correlation functions g(ij)(r) are approximated by their zero-density limit (i.e., the Boltzmann factor), we propose a more accurate representation of g(ij)(r) that takes into account terms up to the first order in the density expansion of the mean-force potential. This improvement is expected to be particularly effective in the case of strong protein-protein interactions at intermediate concentrations. The method is applied to analyze small-angle x-ray scattering data obtained as a function of the ionic strength (from 7 to 507 mM) from acidic solutions of beta-lactoglobulin at the fixed concentration of 10 gl(-1). The results are compared with those obtained using the zero-density approximation and show significant improvement, particularly in the more demanding case of low ionic strength.